Adult stem cells are multipotent cells capable of executing specific differentiation programs in response to injury or key environmental signals. The murine hair follicle undergoes repeated, genetically controlled cycles of proliferation (anagen), regression (catagen) and quiescence (telogen). Intense recent focus has come to elucidate the controls of stem cell quiescence and the timing of re-entering the proliferative anagen stage. Previous studies have shown several key signaling pathways stimulate anagen, such as the Wnt and Sonic hedgehog (Shh) pathways. By contrast, while recent data supports the Bone Morphogenetic Protein (BMP) and calcineurin/NFAT pathways in maintaining quiescence, the higher order mechanisms of cycle timing remain poorly understood. The involvement of calcineurin/NFAT led us to investigate the role of calcium regulatory proteins in hair cycling. A screen for small molecules that topically regulate hair cycling revealed that nifedipine (DHP), an L-type voltage gated calcium channel antagonist, causes precocious entry to anagen in mice. Consistent with this finding, conditional ablation of CACNA1C (CaVI .2), the dominant cutaneous channel, causes similar hair cycling defects and precocious entry to anagen in mice. Further, preliminary data with mice containing a CaVI .2 gain of function mutation that mimics the Timothy syndrome (TS), a human multi-organ system disorder, delays entry into anagen. These data support my hypothesis that calcium transients via the voltage gated calcium channels regulate hair stem cell quiescence through Nfatcl regulation. In order to further dissect the mechanism of calcium signaling and stem cell quiescence, I propose to dissect the molecular defects in of CaVI .2 mutant mice. The goal is to examine the cell lineage requiring CaV1.2 function and to examine alterations in known signaling pathways in mutant mice. We will perform a series of immunofluorescence and X-gal stainings to test our hypothesis. I propose to study calcium signaling in stem cells in vitro and in vivo to test our hypothesis of calcium signaling involved in hair cycling. I will isolate hair follicle stem cells by FACS and culture them in vitro. Using ratiometric calcium dyes and genetically-encoded calcium sensors, I will measure the differences in amount of calcium in stem cells from wild type and Cav1.2 mutant mice. I will take skin samples at different time points over the hair cycle from wild type tissue and mutant tissue to study stem cells in vivo. LAY STATEMENT: Adult stem cells possess the capacity for programmed organ replacement and repair in response to injury or damage. Studying stem cell quiescence is critical because disruption of quiescence leads to cancer. I intend to study the role of calcium signaling in hair follicle stem cell quiescence.